The field of X-ray crystallography involves the determination of the three-dimensional structure of the molecules that make up crystals using bombardment of X-rays and an identification of the crystal structure based on the diffraction of the X-rays by the crystal. Although X-ray crystallographic techniques have been known and used for years, recent advances in this field have allowed for the high-resolution determination of crystal structures to a far greater extent than that which has been previously achievable. In addition, the methodologies that support these studies, including computer graphics, x-ray imaging, computational methods, etc., have all advanced to a level which even furthers the ability of X-ray crystallographic methods to achieve high-resolution pictures of the molecular crystal patterns. In light of the recent advances in this field, X-ray crystallography has proven to be of enormous benefit in applications such as the designing drug-delivery systems (see, e.g., Bugg et al., Scientific American, December 1993, pp. 92-98), and its potential for further breakthroughs in this field is extremely high.
Despite these recent advances in this field, however, one problem that still remains arises because the protein crystals that can be analyzed using state-of-the-art high resolution X-ray crystallography must be grown to a sufficient size in the lab before a high-quality picture of their three-dimensional structure can be achieved. As a result, the high resolution imaging of crystals enabled by the advances in X-ray crystallography is still substantially limited by the inability to successfully grow on a consistent basis high-quality crystals of sufficient size for X-ray analysis. In order for protein crystals to be suitable for structural analysis via X-ray diffraction methods, crystals on the order of about 0.5 mm in diameter or greater must be obtained in order to achieve high resolution of the crystals with diffraction limits of about 3 angstroms or better. This has proved extremely inconvenient and difficult to accomplish on a consistent basis using techniques and crystallization trays known at present.
One of the main techniques available for growing crystals, known as the hanging drop vapor diffusion method, is a method wherein a drop of a solution containing the protein is applied to a glass cover slip and placed upside down in an apparatus such as a vapor diffusion chamber where conditions lead to supersaturation in the protein drop and the initiation of precipitation of the protein crystal. This method is usually troublesome and inefficient because current methods of employing this technique to achieve crystal growth are somewhat primitive, whether conducted manually or through automatic devices, and involve a series of adjustment of the conditions until a suitable experimental regimen is found. In the typical screening methods under this process, it is generally required that the lab technician vary the conditions of pH, buffer type, temperature, protein concentration, precipitant type, precipitant concentration, etc., for each set of experiments, and even adjusting for the myriad of conditions, often only minute samples of the protein can be studied at one time. These variables create an extensive and complex matrix of small experiments, with each experiment requiring another set of protein drops to be affixed to the glass cover slips and inverted and sealed in the vapor pressure chamber. As presently carried out using currently available devices, crystal growth methods such as the hanging drop method are tedious, time-consuming, and hard to carry out successfully and efficiently, and indeed often result in a large percentage of lost crystals and the need to remount the device for additional crystals.
Currently, there are three major sources for vapor diffusion screening trays currently on the market. The first of these screening trays is a conventional 24-chamber culture plate using a tray such as the one disclosed in U.S. Pat. No. 4,495,289 (Lyman et al.), which is generally known as the Linbro plate, and the process using this type of tray involves the tedious steps of inverting and sealing a protein droplet over each individual chamber. This method, which is still most commonly practiced in laboratories growing protein crystals, requires each of the chambers to be sealed tightly using a silicon grease applied to the top of the chamber, and this greasing step causes problems because it is messy and time consuming, and in addition creates problems when the crystal is removed from the greasy cover slip. Another problem resulting from the use of this device arises in that because it operates under conditions of high humidity, associated changes in ambient temperature result in a high percentage of lost runs due to the creation of condensation on the cover slip which dilutes the protein droplet.
The second major vapor equilibration method involves a device such as that depicted in U.S. Pat. No. 5,096,676 (McPherson et al.), which relates to a twenty-four chamber plate generally designed to hold the protein solution in a "sitting-drop" configuration which, unlike hanging-drop trays, allows a protein drop to sit on a central pedestal in the diffusion chamber while the top of the chamber is sealed using thin plastic material such as Mylar. The major problem with this apparatus and method is that the crystallization solutions as used in the McPherson tray are retained in a somewhat deformed plastic pedestal which presents serious limitations with regard to visually screening each experiment, and which interferes with examination of the crystals or other crystalline objects when using polarized light. This is a particular problem because the step involving inspection using polarized light is often the most important part of the screening process in that it is most often used to assess the success of the crystal growing method. Further, when using the McPherson device, the fragile crystals that are grown therein are difficult to see and manipulate because of the angle with which the probes must be manipulated under the microscope, which is necessitated because of the visibility limitations described above with regard to the chamber pedestal of this device. As a result of these drawbacks, the McPherson device results in a greater percentage of lost crystals, which thus causes the user to spend more time and effort in regrowing the crystals for diffraction experiments. Finally, this device also suffers because these trays are more difficult to re-use once each pedestal has been contaminated with protein solution, which often results in additional unwanted microbial growth.
The final method commonly used in encouraging protein crystal growth consists of another twenty-four chamber tray known as the Crystal Plate which is manufactured by Flow Laboratories of McLean, Virginia under license from the American Crystallographic Association. This device is a generally rectangular tray having square chambers which require square cover slips at the top and bottom of each chamber, and in which the reservoir solution is offset from the crystal viewing area. As with most of the prior art devices in this field, the Crystal Plate suffers from drawbacks such as the need to use silicon oil in order to seal both the upper and lower slip covers, which involves the messy and time consuming greasing process which affects the clean and safe removal of the crystal after growth has occurred.
In short, the Crystal Plate and the other prior art devices in this field suffer from a myriad of problems which have limited the use of high-resolution crystallographic techniques in protein analysis. It has thus been highly desirable in light of the recent advances in the field of X-ray crystallography to develop a highly efficient, simple and effective protein crystal growth tray which can be used with a variety of protein growing methods to create protein crystals of sufficient size and stability to be analyzed using X-ray crystallography, and yet which can also avoid the problems associated with the prior art devices.